Induced Pluripotent Stem Cell Meets Severe Combined Immunodeficiency

Severe combined immunodeficiency (SCID) is classified as a primary immunodeficiency, which is characterized by impaired T-lymphocytes differentiation. IL2RG, IL7Ralpha, JAK3, ADA, RAG1/RAG2, and DCLE1C (Artemis) are the most defective genes in SCID. The most recent SCID therapies are based on gene therapy (GT) of hematopoietic stem cells (HSC), which are faced with many challenges. The new studies in the field of stem cells have made great progress in overcoming the challenges ahead. In 2006, Yamanaka et al. achieved "reprogramming" technology by introducing four transcription factors known as Yamanaka factors, which generate induced pluripotent stem cells (iPSC) from somatic cells. It is possible to apply iPSC-derived HSC for transplantation in patients with abnormality or loss of function in specific cells or damaged tissue, such as T-cells and NK-cells in the context of SCID. The iPSC-based HSC transplantation in SCID and other hereditary disorders needs gene correction before transplantation. Furthermore, iPSC technology has been introduced as a promising tool in cellular-molecular disease modeling and drug discovery. In this article, we review iPSC-based GT and modeling for SCID disease and novel approaches of iPSC application in SCID.


Introduction
Severe combined immunodeficiency (SCID) is classified as a primary immunodeficiency (PID), which is characterized by impaired T-lymphocyte differentiation. SCID is a monogenic, heterogeneous, and life-threatening syndrome (1). Considering that both humoral and cellular adaptive immunity are involved, this immunodeficiency is called "combined" because in T -Bphenotypes of SCID, T-cell development, as well as B-cell development is affected. In T -B + phenotypes, the absence of normal T-helpers leads to defective antibody production by normal B-cells. In some subtypes of SCID, the disease can also be accompanied by defective natural killer (NK) cells. These different phenotypes are due to mutations in several genes, which lead to appear in different stages of T-cell development (Fig.1). The worldwide prevalence of SCID is estimated to be in 50,000 to 100,000 of the young population and constitutes 7% of PID patients. Approximately 90% of genetic defects in different forms of SCID have been identified (2,3). The latest therapies regarding SCID are based on gene therapies (Table 1), which so far are faced with many difficulties (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). The new studies in the field of stem cells have made considerable progress in overcoming the challenges ahead.

A review on induced pluripotent stem cell
In 2006, Takahashi et al. (19) achieved "Reprogramming" technology by introducing OCT4, KLF4, SOX2, and C-MYC reprogramming factors (RFs), which are responsible for embryonic-like state, into human fibroblasts. These RFs, known as OKSM factors, generate induced pluripotent stem cells (iPSC) from a somatic cell and reverse its state back into embryonic status, which can later differentiate to various human cells. IPSC-derived pre-differentiated or differentiated cells can be used for transplantation in patients with abnormal or poorly functional specific cell lineage. Considering that harvested cells are autologous, there is no risk of immunological rejection (fully matched HLA-profile) and no concern regarding the low number of transplantable cells. Furthermore, preparing these pluripotent stem cells is a non-invasive method (20).
In addition to other aspects of iPSC-based therapies, there are various studies in the field of cancer and immunodeficiency that led to the creation of iPSC-derived cytotoxic T-lymphocytes (iCTL) and iNKT-Cells, which have major roles in the immune system. The medical applications of iPSC are not limited to cell therapy. Recently, iPSC technology has been introduced as a promising tool for in vitro cellular-molecular disease modeling, drug discovery, and ex-vivo regenerative medicine, including organogenesis and GT (21,22).

The standard severe combined immunodeficiency treatments and challenges
Supportive care, such as intravenous immunoglobulin (IVIG) and antimicrobial agents, may be required for SCID. For example, cotrimoxazole is prescribed as prophylactic agent against P. jirovecii, acyclovir is used for patients with a history of herpes simplex virus infection, and antifungal prophylaxis should also be used. ADA-SCID can also be treated by enzyme replacement therapy (ERT), which consists of ADA coupled to polyethylene glycol (PEG-ADA), but the intramuscular injection is needed at least once a week to eliminate toxic waste. This is a life-saving therapy when other treatments are unavailable or less effective. This treatment is effective in approximately 90% of the patients. However, in some cases, after a period, the dose of PEG-ADA must be increased, mainly when anti-ADA antibodies have been produced. Systemic ERT may also ameliorate hepatic and neurologic dysfunctions in some ADA-SCID patients. A concern regarding ERT is lymphoid and possibly hepatic malignancies and progression of chronic pulmonary insufficiency. ERT before bone marrow transplantation may prevent donor cell engraftment by enhancing endogenous T-cell recovery (23).
Hematopoietic stem cell transplantation (HSCT) is the only definitive treatment for SCID and has more than a 90% chance of success (from geno-identical donors). There are some serious adverse effects, such as graft versus host disease (GvHD) or graft rejection, following the allogenic immune conflict between donor and recipient (24,25). On the other hand, granulocyte colony-stimulating factors (G-CSF), such as filgrastim and lenograstim, which are used to release HSCs into the peripheral blood before leukapheresis, can also be mutagenic. However, chronic infection and thymus problems, which are related to the age of the recipient, can reduce the chance of success or cause damage to specific organs, like liver, kidney, or lung. In NKphenotypes of SCID, myeloablation or immunosuppression is not required for HSCT. NK cells can reduce the survival rate after transplantation of haplo-incompatible HLA. Due to the perpetual B-cell dysfunction, lifelong immunoglobulin substitution may be required to prevent infections caused by inadequate antibody responses (26).
HSCT (10 ,9) Purine Metabolism Transplantation from mismatched donors is still associated with high mortality. GT, as an alternative for haplotype HSCT procedure, is on clinical trials for both ADA-SCID and X-SCID, which will eventually determine the role of GT as a therapeutic option. The first vector that was used in GT was the γ-retroviral vector, which increases the expression of MDS-EVI1 and LMO2 (two insertional hotspots in HSC for γ-retroviral vector) and causes T-cell acute lymphoblastic leukemia (ALL) in some X-linked SCID patients (27)(28)(29). Nevertheless, it has been used in ADA-SCID patients with a mild conditioning regimen, while no genotoxicity was seen. Self-inactivation (SIN) retroviral vector design has deletions in the U3 region of 5ˊLTR beside an internal heterologous promoter, which leads to the reduced incidence or even absence of proto-oncogene activation. Preclinical investigations on retroviral-mediated JAK3 gene transfer shows expression of the exogenous JAK3 protein in animal models. SIN lentiviral vectors (LVs), which are based on the human immunodeficiency virus, have a better safety profile and reduce the risk of insertional mutagenesis. In addition, LVs are superior to γ-retroviral vectors in manipulating human HSCs and maintaining sustained transgene expression. SIN-lentiviral vector GT succeeded in preclinical murine models for ADA-SCID, RAG1-SCID, RAG2-SCID, and Artemis-SCID (30,31). Considering all the challenges and therapeutic potentials of iPSC, the road to treating SCID seems clear, which ultimately leads to shorter and more efficacious treatment courses with fewer side effects.

Induced pluripotent stem cells-based gene therapy meets severe combined immunodeficiency
The invention of iPSC technology has allowed scientists to cure single-gene disorders by creating a healthy cell line from the patient in ex vivo conditions. After a biopsy of healthy cells, they are monitored in the laboratory. Lentiviruses and retroviruses were the first vectors that were used for transferring OKSM factors into somatic cells. The major problem with the use of these vectors is their mutagenicity, but lentiviral vectors are deemed to be less mutagenic than retroviral vectors (32). It has been shown that polycistronic lentiviral viruses, which have been combined with 2A self-cleaving peptides and internal Ribosomal Entry Site (IRES), are sufficient for the integration of RFs. Hence, the recombination of a polycistronic vector system with a lentiviral vector facilitates the clinical applications of iPSC.
There are other methods to generate iPSC. These include non-integrating viruses, such as adenovirus, and non-viral approaches, such as plasmids, DNAdemethylating agents, histone deacethylating agents, and dimethyl transferases (33).
After the gene introduction, a particular time is required to allow the expression of factors and induction of pluripotency. Ultimately, iPSC gains immortality and self-renewal capacity (34). Direct iPSC-based cell therapy is used in non-hereditary diseases. In hereditary disorders, the iPSC-based cell therapy should be combined with GT to correct the defective gene(s). Due to the congenital deficiency in SCID, which is present in HSC, the primary step in iPSC-based treatment is generating "corrected" HSC. Each subtype of SCID results in different molecular mutagenic disorders. The most common ones are listed in Table 2. GT is used to modify these mutations and manipulate normal products by using iPSC-based GT.
There are many targeting vectors when genetically manipulating iPSC. These include classic targeting vectors, DSB-mediated targeting vector, BAC targeting vector, piggyBac targeting vector. Also, helperdependent adenovirus targeting vector, single-strand oligonucleotides (ssODN), and Adeno-associated virus (AAV) targeting vector are among advanced vectors. The nucleases bind to specific sites of DNA and catalyze it to a double-strand break (DSB). In the presence of donor DNA (targeting gene), homologous recombination occurs at a specific genomic site. The CRISPR-Cas9, meganucleases, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) more recently have been engineered for this purpose. They decrease the dysregulation of gene expression and the off-targeting genotoxicity (40)(41)(42)(43). After the correction of mutation, the iPSCs differentiate to HSCs, which is also influenced by specific hematopoietic cytokines and HOXB4 (44). In the next step, SCID patients are treated with induced HSCs transplantation with adequate dosage.  Table 3).

2009
Lei F et al.

Severe combined immunodeficiency modeling based on induced pluripotent stem cells
In addition to all its clinical benefits, iPSC technology is also used in the human disease modeling to identify the exact genomic and molecular pathological pathways of a disorder. It is also used in drug discovery to design efficient, safe, and novel drugs and screen their efficacy and toxicity. The use of animals for human disease modeling have ethical issues, limitation in completely resembling human disease phenotypes due to fundamental differences between human and animal genomes. Furthermore, the inaccessibility to animals and difficult preparation (impossible in some cases) of specific cell-line in vitro makes this method more problematic.
In contrast, patient-derived iPSC has enabled scientists to provide high numbers of disease-specific cell-lines in laboratory conditions and help them overcome the mentioned problems. Moreover, healthy iPS cells, which are derived from the patient, can be used as a control in the modeling process. As mentioned, the accurate recognition of monogenic mutations is a key step in understanding the pathogenesis of the disease (52). Consequently, the action to reverse this mutation is a major step in the treatment of genetic disorders. The in vitro modeling of gene editing is an introduction to the application of in vivo GT. There are conventional tools for gene modification that are used in GT in clinics and modeling. Among the programmable site-specific nucleases, CRISPR-Cas9 system has been highly regarded for its ability to create a wide range of isogenic controls for iPSC-based modeling and its simplicity in design and use (53).
Since the inception of this technology, many studies have been conducted based on iPSC-disease modeling, such as Ciliopathy, Parkinson's disease, hematopoietic abnormalities, cardiac disorders, insulin resistance, and metabolic syndrome, skeletal muscle disorders, schizophrenia pathogenesis, amyotrophic lateral sclerosis, mitochondrial disorders, etc. (54)(55)(56). The use of iPS cells in the modeling of primary immunodeficiencies, such as chronic granulomatous disease (CGD) and SCID, has also been successful (19).
In 2008, one of the first PID models based on iPSC was done by Park et al. (57). In 2015, Chang et al. (46) recognized that the Jak3-deficient T-cell progenitor development is blocked in early stages by using iPSCs derived from SCID patients. In this study, gene editing by the CRISPR-Cas9 system restored the development of early T-cell progenitors. These modified progenitors could differentiate into healthy NK cells and T-cells. In 2016, Brauer et al. (51) modeled T-cell development by iPSCs from RAG1-SCID patients. They recognized that RAG1 mutation has low recombination capability and results in cleavage defects. These studies are powerful tools for identifying the PID mechanisms, pharmacological tests, and GT trials (Fig.2). There are other applications of iPSCs in the field of immunodeficiency (Table 4).

Induced pluripotent stem cell application in secondary diseases of severe combined immunodeficiency and another sight
IPSC can also generate B-cells in T -Bphenotypes of SCID. In advanced SCID, secondary diseases are often seen. In addition to the primary treatment of SCID that restores the immune system, the application of regenerative iPSC technology can also be used for secondary diseases of SCID. Sensorineural hearing loss (SNHL), which is the result of reticular dysgenesis progression, is caused by damage or deficiency in cochlear, which is followed by SCID.
Moreover, deterioration of bone (leading to costochondral dysplasia), thymic epithelium, lung, liver, and brain tissues results in progressive ADA-SCID. PNP-SCID causes neurological abnormalities. RHOH deficiency induces Burkitt lymphoma. ORAI1 and STMI1 deficiencies lead to non-progressive myopathy and ectodermal. MATG1 deficiency can also cause neoplasia (70). Most of the aforementioned disorders are characterized by specific cell-line deficiency. IPSC-derived cells, prepared in vitro, can replace faulty cell-lines. In organopathies, there are clinical approaches to organogenesis and histogenesis based on iPS cells, which were inaccessible before. Malignancies and other disorders are also in the context of iPSC technology. DiGeorge syndrome, characterized by the impaired thymus, leads to SCID. The standard treatment for DiGeorge syndrome is allograft thymus transplantation. The risk of immunological graft rejection can be eliminated by iPSC regenerative technology. Therefore, iPSC appears to be a suitable and comprehensive therapeutic option for SCID patients.
SCID is a fatal PID characterized by impairment in T-cells development. Standard therapeutic plans for SCID are not safe. In grafted cases, there is a risk of GvHD and immune rejection due to the impairment in the immune system. The Side effects of ERT and myeloablation cause a systemic defect in patients. Altogether, we do not have access to optimal therapy for SCID yet.
We discussed different aspects of a critical key in SCID treatment in the future: iPSC therapy, which potentially is optima for SCID therapy. However, many challenges are facing to iPSC technology. As discussed, it could be achieved to the high-pure cell products based on iPSC, including iCTL, iNK cells, HSC, etc., making a safe procedure to transplantation (no immune rejection, no GvHD) as an autologous graft in SCID patients. However, due to the pluripotency state of iPSCs, there is a teratogenesis risk that limited clinical administrations of iPSCs for now. However, there are some reports to overcoming on teratogenesis of iPSCs and entrance in clinical trials. So, iPSC is a promising window for "Bubble boys" in the future, not so far.
Kouchaki et al.   Reproducing the pathognomonic CGD oxidase negative phenotype to show ROS deficiency in neutrophils derived from X-CGD iPSCs, and gp 91phox correction via ZFN and restoration of ROS production in neutrophils.

Modeling
The iPSCs generation from X-CGD and AR47-CGD patients, differentiation to monocytes, and macrophages by a similar cytokine profile to blood-derived macrophages. These macrophages have typical phagocytic properties but lack ROS production. Restoration of NADPH oxidase activity in X-CGD iPSCs after differentiation to neutrophils in two ways: transposonmediated integration of a BAC vector carrying the CYBB gene, and the correction of mutation via homologous recombination.

2017
Brault J et al.

Modeling
The research on enzyme therapy by recombinant NOX2/ p 22phox liposomes in macrophages derived from X-CGD iPSCs that led to the successful delivery of NOX2 and p 22phox to the plasma membrane and regeneration of NADPH oxidase complex and production of superoxide anions.

Conclusion
The technology of iPSC demonstrates a promising future in clinical applications. Since its invention, there is extensive research regarding various reprogramming methods to achieve iPSC. Many researchers have studied its differentiation, application in identifying disease pathogenesis, drug design, histogenesis, organogenesis, and cell therapy. The combination of iPSCs and GT has expanded its therapeutic potential and other aspects of this technology. Most of the clinical applications of iPSC, such as application in SCID, are still in the study phase. Although its introduction to the clinic is not far. As technology is increasingly used by scientists, the treatment of various diseases by iPSC technology is close.